It is widely accepted that Aβ peptide is a causative agent in the development of Alzheimer's disease. Aβ peptides are metabolites of Amyloid-β precursor protein (Alzheimer's disease-associated precursor protein or APP), and consist mainly of 40 to 42 amino acids, Aβ1-40 (“Aβ40”) and Aβ1-42 (“Aβ42”), respectively. Aβ40 and Aβ42 are generated by two enzymatic cleavages occurring close to the C-terminus of APP. The enzymes responsible for the cleavage, the aspartyl protease beta-secretase (“BACE”) and the presenilin-dependent protease γ-secretase (“γ-secretase”), generate the N- and C-termini of Aβ, respectively. The amino terminus of Aβ is formed by β-secretase cleavage between methionine residue 596 and aspartate residue 597 of APP (numbering based on APP 695 isoform). γ-secretase cleaves at varying positions (38-, 40- or 43-residues C-terminal of this β-secretase cleavage product) to release the Aβ peptides. A third enzyme, α-secretase, cleaves the precursor protein between the β- and γ-cleavage sites, thus precluding Aβ production and releasing an approximately 3 kDa peptide known as P3, which is non-pathological. Both β- and α-secretase cleavage also result in soluble, secreted-terminal fragments of APP, known as sAPPβ and sAPPα, respectively. The sAPPα fragment has been suggested to be neuroprotective. These secretases may also be involved in the processing of other important proteins. For example, γ-secretase also cleaves Notch-1 protein.
In normal individuals, the Aβ peptide is found in two predominant forms, the majority Aβ-40 (also known as Aβ1-40 form and the minority Aβ42 (also known as Aβ-42) form, each having a distinct COOH-terminus. The major histological lesions of AD are neuritic plaques and neurofibrillary tangles occurring in affected brain regions. Neuritic plaques consist of AP peptides, primarily Aβ40 and Aβ42. Although healthy neurons produce at least ten times more Aβ40 compared to Aβ42, plaques contain a larger proportion of the less soluble Aβ42. Patients with the most common form of familial Alzheimer's disease show an increase in the amount of the Aβ42 form. The Aβ40 form is not associated with early deposits of amyloid plaques. In contrast, the Aβ42 form accumulates early and predominantly in the parenchymal plaques and there is strong evidence that Aβ42 plays a major role in amyloid plaque deposits in familial Alzheimer's disease patients. Neurofibrillary tangles consist of aggregated tau protein and their role in AD pathology is less clear. AD symptoms are most closely correlated with total brain Aβ rather than plaques. About 10% of AD cases result from autosomal dominant inheritance of mutations in either the APP or the presenilin 1 and presenilin 2 genes. In both cases, increased production of total AP or Aβ42 versus Aβ40 results.
As discussed above, Alzheimer's disease is widely believed to be associated with accumulation of the neurotoxic peptide Aβ. While Aβ is produced by sequential cleavage of APP by BACE and γ-secretase, major efforts to develop selective inhibitors of these enzymes have met with only limited success. For example, most γ-secretase inhibitors suffer from the drawback that they inhibit the cleavage of Notch, a protein essential for normal development.
In addition to BACE and γ-secretase, Casein Kinase 1 (“CK1”) has also been implicated in the production of Aβ-40 and Aβ-42 peptides. For example, CK1δ□ mRNA has been shown to be up-regulated in AD brain samples (Yasojima, K. et al. (2000) Brain Res 865, 116-20) and may be associated with a pathological association with tau (Schwab, C. et al., (2000) Neurobiol Aging 21, 503-10). Interestingly, Glycogen Synthase Kinase 3 (GSK-3), one of the most studied kinases in the Alzheimer field, can phosphorylate its substrates only if they are pre-phosphorylated by a priming kinase, and CK1 is one of the few GSK-3 priming kinases (PKA, CK1, CK2, Cdk5 and DYRK1A) (See e.g., Meijer, L. et al., (2004) Trends Pharmacol Sci 25, 471-80). It has also been shown that CK1 is an upstream regulator of Cdk5, another protein kinase implicated in AD (Liu, F. et al., (2001) Proc Natl Acad Sci USA 98, 11062-8). Moreover, CK1 phosphorylates BACE, and regulates its subcellular location (Pastorino, L., Ikin, A. F., Nairn, A. C., Pursnani, A. & Buxbaum, J. D. (2002) Mol Cell Neurosci 19, 175-85.). CK1 has also been shown to phosphorylate PS2 (Walter, J., Grunberg, J., Schindzielorz, A. & Haass, C. (1998) Biochemistry 37, 5961-7.
In mice, CK1 consists of a family of eight genes that appear to function as monomeric enzymes: α, γ1, γ2, γ3, δ, ε1, ε2 and ε3. Family members contain a highly conserved 290 residue N-terminal catalytic domain coupled to a variable C-terminal region that ranges in size from 40 to 180 amino acids. It is possible that the different isoforms are expressed in different neuronal populations and/or are targeted to different regions of the neuron, and thus may have access to different substrates. Little is known about the regulation of CK1. CK1 is basally active but certain isoforms (particularly CK1δ and ε) are regulated by inhibitory autophosphorylation at their C-terminal regions (Zhai, L. et al., (1995) J Biol Chem 270, 12717-24). Notably, it has been shown that the C-terminal region of CK1ε is phosphorylated at multiple sites and that enzyme activity can be increased following dephosphorylation by a signaling pathway involving activation of the serine/threonine phosphatase, calcineurin in response to stimulation of metabotropic glutamate receptors in neurons (Liu, F., et al., (2001) Proc Natl Acad Sci U SA 98, 11062-8; Liu, S. J. et al., (2003) J Neurochem 87, 1333-44).
CK1 is localized to both the cytosol and the nucleus; the C-terminal region of CK1 has been shown to promote differential subcellular localization of individual isoforms (e.g. nucleus versus cytoplasm). A number of proteins have been found to interact with CK1 isoforms in non-neuronal tissues resulting in its targeting to specific signaling pathways (Amit, S., et al. (2002) Genes Dev 16, 1066-76; Cong, F., et al., (2004) Mol Cell Biol 24, 2000-11; Davidson, G., et al., (2005) Nature 438, 867-72).
Association of CK1 with the plasma membrane and the cytoskeleton has also been reported. (Ahmed, K. (1994) Cell Mol Biol Res 40, 1-11; Vancura, A. et al., (1994) J Biol Chem 269, 19271-8; Walter, J., Schnolzer, M., Pyerin, W., Kinzel, V. & Kubler, D. (1996) J Biol Chem 271, 111-9). In neurons, CK1 phosphorylates a variety of proteins including transcriptional factors, as well as certain synaptic vesicle proteins (Issinger, 0. G. (1993) Pharmacol Ther 59, 1-30; Gross, S. D. et al. (1995) J Cell Biol 130, 711-24). CK1δ and CK1ε, being predominantly expressed in brain (Gross, S. D. & Anderson, R. A. (1998) Cell Signal 10, 699-711), have been implicated in several important brain processes, including but not limited to: dopamine signaling (DARPP-32 phosphorylation), circadian rhythm (mPer phosphorylation) and brain receptor signaling (Desdouits, F., et al., (1995) Proc Natl Acad Sci USA 92, 2682-5; Kloss, B., et al. (2001) Neuron 30, 699-706; Singh, T. J. et al. (1995) FEBS Lett 358, 267-72.